DE102004056794A1 - Protein biochip for the validation of binders - Google Patents

Abstract

The present invention relates to an array of proteins containing at least one cDNA expression library and its use as a protein biochip, in particular for the validation of binders and protein binders, and to methods for the simultaneous determination of quantitative quantities.

Description

The The present invention relates to an assembly of proteins at least one cDNA expression library and its use as a protein biochip, in particular for validation of binders in the presence of protein binders and simultaneous methods Determination of quantitative quantities.

Protein biochips gain an increasing industrial importance in analytics and diagnostics as well as in pharmaceutical development.

Especially could with the help of protein biochips in the analysis of the genome and gene expression a big one Information gain be made. Here is the fast and highly parallel detection of a large number of specifically binding analysis molecules in one single experiment allows. To produce protein biochips it is necessary to obtain the required proteins to disposal to have. Protein expression libraries have been established for this purpose. The high-throughput cloning of defined open reading frames is a possibility (Heyman, J.A., Cornthwaite, J., Foncerrada, L., Gilmore, J.R., Gontang, E., Hartman, K.J., Hernandez, C.L., Hood, R., Hull, H.M., Lee, W.Y., Marcil, R., Marsh, E.J., Mudd, K.M., Patino, M.J., Purcell, T.J., Rowland, J.J., Sindici, M.L. and Hoeffler, J.P. (1999) Genome scale cloning and expression of individual open reading frames using topoisomerase I-mediated ligation. Genome Res, 9, 383-392; Kersten, B., Feilner, T., Kramer, A., Wehrmeyer, S., Possling, A., Witt, I., Zanor, M.I., Stracke, R., Lueking, A., Kreutzberger, J., Lehrach, H. and Cahill, D.J. (2003) Generation. of Arabidopsis protein chip for antibody and serum screening. Plant Molecular Biology, 52, 999-1010; Reboul, J., Vaglio, P., Rual, J.F., Lamesch, P., Martinez, M., Armstrong, C.M., Li, S., Jacotot, L., Bertin, N., Janky, R., Moore, T., Hudson, J.R., Jr., Hartley, J.L., Brasch, M.A., Vandenhaute, J., Boulton, S., Endress, G.A., Jenna, S., Chevet, E., Papasotiropoulos, V., Tolias, P.P., Ptacek, J., Snyder, M., Huang, R., Chance, M.R., Lee, H., Doucette strain, L., Hill, D.E. and Vidal, M. (2003) C. elegans ORFeome version 1.1: experimental verification of the genome annotation and resource for proteome-scale protein expression. Nat Genet, 34, 35-41 .; Walhout, A.J., Temple, G.F., Brasch, M.A., Hartley, J.L., Lorson, M.A., van den Heuvel, S. and Vidal, M. (2000) GATEWAY recombinational cloning: application to the cloning of large numbers of open reading frames or ORFeoms. Methods Enzymol, 328, 575-592). However, one depends Such an approach strongly correlates with the progress of genome sequencing projects and the annotation of these gene sequences together. Furthermore is the determination of the expressed sequence due to differential Splicing operations are not always unique. This problem can be overcome by the application of cDNA expression libraries be bypassed (Büssow, K., Cahill, D., Nietfeld, W., Bancroft, D., Scherzinger, E., Lehrach, H. and Walter, G. (1998) A method for global protein expression and antibody screening on high-density filters of an arrayed cDNA library. Nucleic Acids Research, 26, 5007-5008; Büssow, K., Nordhoff, E., Lübbert, C., Lehrach, H. and Walter, G. (2000) A human cDNA library for high-throughput protein expression screening. Genomics, 65, 1-8; Wood, C., Lueking, A., Bovekamp, L., Gutjahr, C., Bolotina, N., Lehrach, H. and Cahill, D.J. (2001) A human cDNA expression library in yeast enriched for open reading frames. Genome Res, 11, 1730-1735; Lueking, A., Wood, C., Gotthold, C., Lehrach, H. and Cahill, D. (2000) A system for dual protein expression in Pichia pastoris and Escherichia coli, protein Expr. Purif., 20, 372-378). In this case, the cDNA of a particular tissue in a bacterial or a yeast expression vector cloned. The expression The vectors used are generally characterized in that they carry inducible promoters that coincide with the timing controlling protein expression. About that In addition, expression vectors have sequences for so-called affinity epitopes or proteins, which on the one hand the specific proof of recombinant fusion proteins by means of one against the affinity epitope directed antibody secondly, the specific purification is via affinity chromatography (IMAC).

For example were the gene products of a cDNA expression library from human fetal brain tissue in the bacterial expression system Escherichia coli in high-density format on a Membrane arranged and could successfully with different antibodies be screened. It could be shown that the proportion of full-length proteins is at least 66%. The recombinant proteins of this library could about it Be expressed in high throughput and purified brown P., Hu, Y., Shen, B., Halleck, A., Koundinya, M., Harlow, E. and LaBaer, J. (2002) Proteome scale purification of human proteins from bacteria. Proc Natl Acad Sci U S A, 99, 2654-2659; Büssow (2000) supra; Lueking, A., Horn, M., Eickhoff, H., Büssow, K., Lehrach, H. and Walter, G. (1999) Protein microarrays for genes expression and antibody screening. Analytical Biochemistry, 270, 103-111. Such protein biochips based on cDNA expression libraries are in particular Subject of WO 99/57311 and WO 99/57312.

in the Prior art are such arrangements of proteins on the Base of cDNA expression libraries mainly for qualitative analysis been used. However, there is a great need for such Protein biochips based on cDNA expression libraries for the validation of binders on protein binders (e.g. Antibody / antigen).

Especially are for the quality determination a binder to a protein binder quantitative sizes of Relevance such as "cross-reactivity", "specificity" or "sensitivity", "dynamic range".

in the In the prior art, such quantitative quantities are not parallel or simultaneous, but individually and temporally offset preferably with conventional Methods, such as Dot blot, Western blot, ELISA, EIA or RIA. Also have been recently Protein arrays and protein biochips used. For example, could in single measurement the binding specificity of different monoclonal antibody like anti-HSP90β, anti-GAPDH or anti-α-tubulin on a protein microarray, consisting of 96 human recombinantly expressed proteins (Lueking (1999) supra). In addition, the cross-reactivity of two monoclonal antibody against about 2500 different proteins are investigated (Lueking, A., Possling, A., Huber, O., Beveridge, A., Horn, M., Eickhoff, H., Schuchardt, J., Lehrach, H. and Cahill, D.J. (2003) A Nonredundant Human Protein Chip for Antibody Screening and Serum Profiling. Mol Cell Proteomics, 2, 1342 to 1349).

Therefore The invention relates to a protein biochip or a protein microarray with the task at least two or more quantitative sizes in parallel or to be able to determine simultaneously or simultaneously.

The Task is solved by that an array of protein binders containing at least one cDNA expression library is provided, wherein a first region of the arrangement a Totality of protein binders, if necessary represented together with binders, wherein in a second region of the array at least one selected protein binder from the totality of protein binders represented in different quantities relative to one unit is.

Of the Term "protein binder" in the sense of this Invention means that a binder in the presence of a protein binder contacted or binds to this or at least in interaction occurs. In the broadest sense, the binder is addressed to the protein binder or the binder recognizes the protein binder or the protein binder has the Potential to interact with a binder.

Protein Binder can Proteins, peptides, modified proteins / peptides, recombinant proteins / peptides, antibody or antigens, or other proteins that represents a protein biochip according to the invention can be. Ideally, the protein binders are obtained from the cDNA expression library and are immobilized accordingly. Furthermore, the protein binders can be chemically or be physically modified, e.g. not conclusive: biotinylation, Phosphorylation, denatured via Heat or denatured by 4-8 molar urea, mercaptoethanol treatment for reduction of hydrogen sulfide bridges.

suitable Binders in the sense of this invention can not be conclusive: Proteins, peptides, modified proteins / peptides, recombinant proteins / peptides, antibody or antigens, or other proteins, proteids, hormones, non-proteins such as. RNA, DNA or aptamers, chemical probes, chemical molecules, substance libraries, Pharmaceuticals etc.

The Binder can in (on) purified form and mixed or even in a heterogeneous Protein mixture, such as a lysate or digest (e.g., bacterial lysate, mammalian cell lysate) available. This reflects the quality of the binder in complex Mixtures, such as occur in immunohistochemistry, contrary.

In a further embodiment In the invention, the binder is also in the entirety of the protein binders (first portion of the inventive arrangement) (e.g. immobilized). Particularly advantageous, the quantification of specific Signals, as well as cross-reacting signals are performed. this makes possible a direct comparison between the protein binders. Especially an internal standard is made possible.

In particular, the invention relates to such an arrangement of protein binders in which the first region and the second region form a unit, this unit being accessible to at least one binder is. In particular, this unit is a physical and spatial entity. A possible embodiment shows 1 (Content = area).

Especially can be advantageous the embodiments of the invention for the determination of quantitative quantities, such as "cross-reactivity" and "sensitivity" and "dynamic" Area "the binder or protein binders.

in the Meaning of this invention means "cross-reactivity", the property the protein binder, in addition to actual Bind also to heterologous, similar Structures (e.g., other similar ones Binder) bind or at least interact with them to step. For the quality a protein binder is a low cross-reactivity prevail crucial. In other words, the more clearly a binder for one Protein binder recognizes or is addressed, the more specific and more valuable is the binder.

The "degree of Cross-reactivity "and the" specificity "are therefore closely related connected. Binders that show a high degree of cross-reactivity are considered nonspecific classified. About that In addition, the specificity also be determined by the fact that the binder Isoforms or processed forms (for example phosphorylation) recognize and / or differentiate from protein binders.

The "sensitivity" of a binder in the sense of this invention describes the minimum required concentration of the protein binder, which can still be detected with this binder (see 2a ).

For the purposes of this invention, the "dynamic range" describes the range between the highest and lowest detection value (signal intensity) between binder / protein binders, whereby a linear relationship between signal intensity and concentration of the detected protein binder usually exists. plotted as signal intensities versus amounts per unit, the correlation in which the binder binds to the protein binder (see 2a ).

Therefore also relates to such an arrangement of protein binders Diagnostic or a protein biochip or protein microarray. In the context of this invention, "arrangement" synonymously means "array" and if this "array" for the identification of binders or Protein binders is used, this is an "assay" to understand a preferred embodiment the arrangement is such that those represented on the arrangement Protein binder in Form of a grid. Furthermore, such arrangements are preferred a high-density array of protein binders allow. Such high-density arrangements are for example in the WO 99/57311 and WO 99/57312.

In a particular embodiment the protein binders are clones, especially in the first region the arrangement according to the invention, in front. Such clones can be obtained for example by means of a cDNA expression library according to the invention (Buessow et al. 1998 (supra)). In a preferred embodiment, such expression libraries become containing clones by means of expression vectors from an expressing cDNA library obtained. These expression vectors preferably contain inducible promoters. Induction of expression may be e.g. by means of an inductor, such as IPTG. Suitable expression vectors are described in Terpe et al. (Terpe T Appl Microbiol Biotechnol. 2003 Jan; 60 (5): 523-33). additionally the expression product is preferably in the form of a fusion protein which is, for example, at least one affinity epitope or "day" contains. Of the Day can be such as c-myc, His-tag, Arg-tag, FLAG, alkaline phosphatase, V5 tag, T7 tag or strep tag, HAT tag, NusA, S-tag, SBP tag, Thioredoxin, DsbA, a fusion protein, preferably a cellulose-binding one Domain, green fluorescent Protein, maltose binding protein, calmodulin binding protein, Glutathione S-transferase or lacZ included.

expression libraries are known in the art, these can according to standard works, such as Sambrook et al, "Molecular Cloning, A laboratory handbook, 2nd edition (1989), CSH press, Cold Spring Harbor, New York. Further preferred are such expression libraries that are tissue specific (e.g. human tissue, in particular human organs). Furthermore, according to the invention also such expression libraries included by means of exontrapping can be obtained. Instead of expression library can be synonymous of an expression library to be spoken.

Also preferred are protein biochips or corresponding expression libraries that have no redundancy (called: UNIclone ^® library) and can be prepared according to the teachings of WO 99/57311 and WO 99/57312, for example. These preferred Uniclone libraries have a high content of non-defective, fully expressed proteins of a cDNA expression library.

in the Within the scope of this invention the clones, too, will not be as final as transformed Bacteria, recombinant phages or transformed cells of mammals, insects, Mushrooms, yeasts or plants.

The Clones become a solid carrier fixed or immobilized.

Of the Term "solid Carrier "includes versions like a filter, a membrane, a magnetic bead, a silicon wafer, glass, metal, a chip, a mass spectrometric Target or a matrix.

When Filter is PVDF or nylon preferred (e.g., Hybond N + Amersham).

In a further preferred embodiment the inventive arrangement This corresponds to a grid that the order of a microtiter plate (96 wells, 384 wells or more), a silicon wafer, a chip, a mass spectrometric target or a matrix.

Allowed according to the invention Such an inventive arrangement a screening of at least one binder on the protein binders with a subsequent one Evaluation.

After this the binder contacts a protein binder, the evaluation takes place, for example, using commercially available image analysis software (GenePix Pro (Axon Laboratories), Aida (Raytest), ScanArray (Packard Bioscience).

to Evaluation for the purpose of determining the sensitivity and the dynamic range are the signal intensities on the Applied concentrations.

to Determination of cross-reactivity becomes the signal intensity the specific protein binder (e.g., antigen) against which the binders are set to 100%. The signal intensities of the cross-reacting protein binders be in proportion to this set and used to evaluate the binder.

Therefore allows the arrangement according to the invention the simultaneous or simultaneous determination of quantitative quantities.

This is in particular justified in that that the assembly is a sample inividation of the binder for the first and second area of the arrangement according to the invention allowed. Of the first and second area is accessible a sample containing at least one binder. This leads to a advantageous high reproducibility and standardization of Samples and allows the high-throughput application of this arrangement according to the invention.

The Visualization of such binders can be carried out, for example, by means of fluorescence marking, Biotinization or radio-isotope labeling in the usual way. One Readout is e.g. by means of a microarray laser scanner.

Of the Term "different Quantities in terms of a (local) unit "means that, for example, different Concentrations (weight / unit area, Weight / volume unit, mol (particle number) / unit area, mol (particle number) / volume unit) available. In particular, a so-called linear concentration series or linear dilution series available. Preferred series are 1 μmol to 0.1 Amol, in particular 100 pmol to 0.1 fmol, more preferably 10 pmol to 1 fmol a unit area or volume unit or per spot (eg a microarray).

A "whole of protein binders " in the sense of the invention that a sufficient number of different Protein binders and, if necessary, binders can be used. Prefers are at least 96 to 25000 (numerical) or more from different Protein binders. Preferably, however, more than 2500, more preferably 10,000 or more protein binders, for example, from an expression library result.

Further allows the arrangement according to the invention of protein binders the simultaneous / simultaneous validation of multiple binders or their comparative analysis in terms of quantitative sizes.

Therefore, the invention relates to a method wherein the unit from the first and second region is contacted with at least one binder. The invention also relates to a method for comparing two or more binders to protein binders, wherein an inventive arrangement of protein binders is brought into contact with the binders to be compared and evaluated.

For the rating the quality (Validation) of the protein binder becomes the signal intensity a specific binder against which the binders are directed, set to 100. The signal intensities of the cross-reacting binder / protein binders become this in proportion set and used to evaluate the binder.

Table 1: Examination of cross-reactivity. The example of three binders (A, B, C) shows the different cross-reactivity.

For example, the analysis of three different binders (see 2 B ) against a protein binder (eg, antigen) have different degrees of cross-reactivity. Binder A and C show cross-reactivity to three other proteins respectively (corresponds to 3.1% total cross-reactivity), while Binder B has no cross-reactivity. In a second step one would like to determine the strength (in%) of the crossreactively reacting proteins in comparison to the specifically reacting antigen. For this purpose, the signal intensity of the specific antigens is equal to 100 (Binder A: 62,000 signal intensity, Binder C: 900 signal intensity), and the signal intensities of the cross-reacting proteins are related to this. For Binder A, cross-reacting signals of 0.7% (435 signal intensity), 1.02% (631), and 6.2% (3858) are obtained. For Binder C, cross-reacting signals of 58% (523 signal intensity), 119% (1077) and 144% (1301) are obtained (see Table 1).

The The following examples serve to illustrate the invention without to limit the invention to these.

Execution:

Generation of proteins for content 2 field:

It the culture of the expression clones is carried out from a cDNA expression library (Buessow (1998) supra) in high-throughput format (96-well microtiter plate) overnight. Protein expression of the clones is induced the next day, followed from protein purification in high throughput format. This entire Process is described in the following publications: (Büssow (2000) supra, Lueking (1999) supra, Lueking (2003) supra). The cleaned up Proteins will be quality-controlled via SDS-PAGE subjected.

Come Proteins of the Content 1 field also a cDNA expression library as she did under Büssow et al., 1998 (supra), they can be expressed analogously and purified. Otherwise, these proteins can also have a other origin (e.g., commercially available).

Generation of the "binder Validation Chip ":

First, a suitable carrier is selected. This may be different coated microscope slides, which are available from various commercial suppliers (eg, Schleicher and Schuell, Menzel, Sigma, etc.). Both two- and three-dimensional surfaces can be used well. Thereafter, the concentrations of the specific antigens / proteins are determined and related to the molarity so that an indication of "x" pmol / μl per antigen can be made, based on which these specific antigens / proteins are expressed in defined concentrations (from high to low in the range of 10 pmol / μl to 1 fmol / μl). The proteins of Content 1 and 2 and their dilutions are stored in a 384 microtiter plate and transferred to the selected surface using a standard spotting robot (as used for the production of DNA chips) (eg described in Lueking 2003 (supra)).

After that are the loaded protein biochips for the planned incubations ready.

incubation:

to Visualization will be against fluorescence-labeled secondary antibodies the binder eingsetzt, or the binder is already floureszenz-marked in front.

The usual Steps are in Lueking et al. 1999 (supra), Lueking et al. 2003 (supra)), the specific incubation conditions (Blocking Buffer, wash buffer, secondary Antibody, Incubation times) become more common Way given by the binder.

visualization:

With commercial Microarray laser scanners (ScanArray 4000 (Packard Bioscience)).

Image analysis:

With commercial Image analysis software GenePix Pro (Axon Laboratories), Aida (Raytest), ScanArray (Packard Bioscience).

Evaluation:

evaluation occurs e.g. using Excel (Microsoft), whereby for the determination of the sensitivity and the dynamic Range the signal intensities over the Concentration are applied.

to Determination of cross-reactivity becomes the signal intensity the specific antigens against which the binders are directed, set to 100%. The signal intensities of the cross-reacting antigens / proteins become in relation to this set and used to evaluate the binder.

Description of the figures:

1 : Representation of the binder validation chip. This protein biochip consists of two areas. In the first area (Content 1), the protein binders to be investigated are immobilized in specific concentrations. The second region (Coontent 2) contains a large number of different protein binders, which can be used to determine the degree of cross-reactivity.

2a : Determination of Sensitivity and Dynamic Range. If the signal intensities are plotted against the concentration of a protein binder, a statement about the dynamic range can be made by the resulting curve. The smallest measurable signal intensity (above the background noise) determines the minimum measurable concentration and thus the sensitivity of the binder.

2 B : Determination of the sensitivity and the dynamic range using the example of three binders against one antigen. For this purpose, the signal intensities of three different binders were plotted versus the concentration of a protein binder. Different dynamic ranges and bonding behavior can be determined for the three binders.

Claims (19)

Arrangement of protein binders containing at least a cDNA expression library, being a first area of the Arrangement represents a set of protein binders, in which in a second region of the array, at least one selected protein binder from the totality of protein binders in different amounts represented by a unit is. Arrangement of protein binders according to claim 1, characterized characterized in that the binder is also in the totality of Represents protein binders is. Arrangement of protein binders according to claim 1 or 2, characterized in that the first area and the second Forming a unit. Arrangement according to claim 3, characterized that the unit is accessible to at least one binder. Arrangement according to one of Claims 1 to 4, characterized that the first range is a total of at least 96 to 25,000 or more protein binder and are arranged in the form of a grid, in particular the order of magnitude a microtiter plate, a silicon wafer, a chip, a mass spectrometric Has targets or a matrix. Arrangement according to one of claims 1 to 5, characterized that the protein binders are selected are from proteins, peptides, modified proteins / peptides, recombinant Proteins / peptides, antibodies or Antigens. Arrangement according to one of claims 1 to 6, characterized that the protein binders are chemically or physically modified. Arrangement according to one of claims 1 to 7, characterized that the protein binders are clones of a cDNA expression library, in particular transformed bacteria, recombinant phages or transformed Mammalian cells, Insects, fungi, yeasts or plants. Arrangement according to claims 1 to 8, characterized the cDNA expression library is tissue-specific. Arrangement according to one of claims 1 to 9, characterized that the cDNA expression library by means of Expressionvoren from are obtained from an expression library. Arrangement according to claim 10, characterized the expression vectors contain inducible promoters. Arrangement according to one of claims 1 to 11, characterized that the protein binders are present as fusion proteins, in particular an affinity epitope or a day such as c-myc, his-tag, arg-tag, FLAG, alkaline Phosphatase, V5 tag, T7 tag or strep tag, HAT tag, NusA, S tag, SBP-tag, thioredoxin, DsbA, a fusion protein, preferably one Cellulose-binding domain, green fluorescent protein, Maltose binding protein, calmodulin binding protein, glutathione S-transferase or lacZ included. Arrangement according to one of Claims 1 to 12, characterized that the different amounts relative to one unit in the second Area of arrangement in the form of a linear concentration series or linear dilution series available. Arrangement according to one of claims 1 to 11, characterized that the arrangement for simultaneous or simultaneous determination qualitative sizes of Binder or protein binder serves. Arrangement according to claim 14, characterized that the qualitative parameters are cross-reactivity or specificity and sensitivity as well are dynamic range. Arrangement according to one of Claims 1 to 4, characterized that the binder is selected from proteins, peptides, modified proteins / peptides, recombinant Proteins / peptides, antibodies or antigens, proteids, hormones, RNA, DNA or aptamers, chemical Probes, chemical molecules, Substance libraries, pharmaceuticals. Arrangement according to one of claims 1 to 4 and 14, characterized characterized in that the binders are mixed in (on) purified form as well or in a heterogeneous protein mixture, such as a lysate or digest. Protein biochip or protein microarray from an arrangement according to one of claims 1 to 17. Method for comparing two or more binders to protein binders, wherein an arrangement according to claims 1 to 18 with the binders to be compared with the protein binders in Contact is brought.